This invention is directed to peptides related to somatostatin and to methods for pharmaceutical treatment of mammals using such peptides. More specifically, the invention relates to shortened receptor-selective somatostatin analogs and the inclusion of an amino acid substitution in such analogs that confers receptor-selectivity thereto, to pharmaceutical compositions containing such peptides, to such peptides complexed with radioactive nuclides or conjugated to cytotoxins, to methods of diagnostic and therapeutic treatment of mammals using such peptides, particularly peptides that are chelated or otherwise labelled, and to methods for screening for more effective drugs using such peptides.
The cyclic tetradecapeptide somatostatin-14 (SRIF) was originally isolated from the hypothalamus and characterized as a physiological inhibitor of growth hormone release from the anterior pituitary. It was characterized by Guillemin et al. and is described in U.S. Pat. No. 3,904,594 (Sep. 9, 1975). This tetradecapeptide has a bridging or cyclizing bond between the sulfhydryl groups of the two cysteinyl amino acid residues in the 3- and 14-positions. SRIF was found to also regulate insulin, glucagon and amylase secretion from the pancreas, and gastric acid release in the stomach, e.g. it inhibits the effects of pentagastrin and histamine on the gastric mucosa. SRIF is also expressed in intrahypothalamic regions of the brain and has a role in the regulation of locomotor activity and cognitive functions. SRIF is localized throughout the central nervous system, where it acts as a neurotransmitter. In the central nervous system, SRIF has been shown to both positively and negatively regulate neuronal firing, to affect the release of other neurotransmitters, and to modulate motor activity and cognitive processes.
Somatostatin and many analogs of somatostatin exhibit activity in respect to the inhibition of growth hormone (GH) secretion from cultured, dispersed rat anterior pituitary cells in vitro; they also inhibit GH, insulin and glucagon secretion in vivo in the rat and in other mammals. One such analog is [D-Trp8]-SRIF which has the amino acid sequence: (cyclo 3-14)H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH, which is disclosed in U.S. Pat. No. 4,372,884 (Feb. 8, 1983). Somatostatin has also been found to inhibit the secretion of gastrin and secretin by acting directly upon the secretory elements of the stomach and pancreas, respectively, and somatostatin is being sold commercially in Europe for the treatment of ulcer patients. The powerful inhibitory effects of somatostatin on the secretion not only of GH but also of insulin and glucagon have led to studies of a possible role of somatostatin in the management or treatment of juvenile diabetes and have proved useful in studying the physiological and pathological effects of these hormones on human metabolism. SRIF is also known to inhibit the growth of certain tumors.
L. Pradayrol, et al. in FEBS Letters 109, January 1980, pages 55-58, reported the isolation and characterization of somatostatin-28 (SRIF-28) from porcine upper small intestine. SRIF-28 is an N-terminally extended version of SRIF which has an additional 14 amino acid residues and which shows some increased potency when administered in vivo.
SRIF affects multiple cellular processes. Studies have shown that SRIF is an inhibitory regulator of adenylyl cyclase in different tissues. SRIF also regulates the conductance of ionic channels, including both K+ and Ca2+ channels. These actions of SRIF are mediated via pertussis toxin-sensitive guanine nucleotide-binding proteins. SRIF also regulates the activity of tyrosine phosphatases, the Na+/H+ antiport, and cellular proliferation through pertussis toxin-insensitive mechanisms.
SRIF induces its biological effects by interacting with a family of membrane-bound structurally similar receptors. Five SRIF receptors have been cloned and are referred to as SSTR1-5. Human SSTR1, mouse SSTR2 and mouse SSTR3 are described in Raynor et al., Molecular Pharmacology, 43, 838-844 (1993), and all five human SRIF receptors are now available for research purposes. Human SSTR1, 2 and 3 are also disclosed in U.S. Pat. No. 5,436,155 (Jul. 25, 1995). Additional SRIF receptors are disclosed in U.S. Pat. No. 5,668,006 (Sep. 16, 1997) and 5,929,209 (Jul. 27, 1999). All five receptors bind SRIF and SRIF-28 with high affinity. Selective agonists at SSTR2 and SSTR5 have been identified and used to reveal distinct functions of these receptors. These two receptors are believed to be the predominant subtypes in peripheral tissues. SSTR2 is believed to mediate the inhibition of growth hormone, glucagon and gastric acid secretion. In contrast, SSTR5 appears to be primarily involved in the control of insulin and amylase release. SSTR3 is found in cortex tissue, in the pituitary and in ademoma tumor tissue; it is believed to mediate inhibition of gastric smooth muscle contraction upon binding by SRIF. These findings indicate that different receptor subtypes mediate distinct functions of SRIF in the body.
There are different types of tissues in the human body that express somatostatin receptors including: (1) the gastrointestinal tract, likely including the mucosa and smooth muscle, (2) the peripheral nervous system, (3) the endocrine system, (4) the vascular system and (5) lymphoid tissue, where the receptors are preferentially located in germinal centers. In all these cases, somatostatin binding is of high affinity and specific for bioactive somatostatin analogs.
Somatostatin receptors are also expressed in pathological states, particularly in neuroendocrine tumors of the gastrointestinal tract. Most human tumors originating from the somatostatin target tissue have conserved their somatostatin receptors. It was first observed in growth hormone producing adenomas and TSH-producing adenomas; about one-half of endocrine inactive adenomas display somatostatin receptors. Ninety percent of the cardinoids and a majority of islet-cell carcinomas, including their metastasis, usually have a high density of somatostatin receptors. However, only 10 percent of colorectal carcinomas and none of the exocrine pancreatic carcinomas contain somatostatin receptors. The somatostatin receptors in tumors can be identified using in vitro binding methods or using in vivo imaging techniques; the latter allow the precise localization of the tumors and their metastasis in the patients. Because somatostatin receptors in gastroenteropancreatic tumors are functional, their identification can be used to assess the therapeutic efficacy of an analog to inhibit excessive hormone release in the patients.
A cyclic SRIF analog, variously termed SMS-201-995 and Octreotide, i.e. D-Phe-c[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-ol is being used clinically to inhibit certain tumor growth; analogs complexed with 111In or the like are also used as diagnostic agents to detect SRIF receptors expressed in cancers. Two similar octapeptide analogs having 6-membered rings, i.e. Lanreotide and Vapreotide, have also been developed, see Smith-Jones et al., Endocrinology, 140, 5136-5148 (1999). A number of versions of these somatostatin analogs have been developed for use in radioimaging or as radiopharmaceuticals in radionuclide therapy. For radioimaging, for example, labeling with 123I can be used as disclosed in U.K. Patent Application 8927255.3 and as described in Bakker et al., 1991, J. Nucl. Med., 32:1184-1189. Proteins have been previously radiolabeled through the use of chelating agents, and there are various examples of complexing somatostatin analogs with 99Tc, 90Y or 111In, see U.S. Pat. Nos. 5,620,675 and 5,716,596. A variety of complexing agents have been used including DTPA (Dirgolini, et al., European Journal of Nuclear Medicine, 23:1388-1399, October 1996); (Stabin, et al., J. Nuc. Med., 38:1919-1922, December 1997); (Vallabhajosula, et al., J. Nuc. Med., 37:1016-1022, June 1996); DOTA (De Jong, et al., Int. J. Cancer, 75:406-411, 1998); (Froidevaux, et al., Peptide Science-Present and Future, 670-673, 1999); HYNIC (Decristoforo, et al. Eur. J. Nuc. Med., 26:869-876); (Krois, et al., Liebigs Ann., 1463-1469, 1996); and P2S2-COOH (Karra, et al., Bioconjugate Chem., 10:254-260, 1999. U.S. Pat. No. 5,597,894 discloses analogs of Octreotide modified to facilitate radiolabeling.
Octreotide and other clinically used SRIF analogs interact significantly with three of the receptor subtypes, i.e. SSTR2, SSTR3 and SSTR5. SSTR2 and SSTR5 have recently been reported to mediate antiproliferative effects of SRIF on tumor cell growth; therefore, they may mediate the clinical effects of Octreotide in humans. U.S. Pat. No. 5,750,499 (May 12, 1998) discloses SRIF analogs which are selective for SSTR1. A recent comprehensive review of SRIF and its receptors is found in Patel, Y. C. xe2x80x9cSomatostatin and its receptor familyxe2x80x9d, Front. Neuroendocrinol, 1999, 20, 157-198.
SSTR3 was one of the first SRIF receptors cloned. It has high affinity for SRIF and SRIF-28 but low affinity for most synthetic analogs of SRIF. In a variety of human tumors, SSTR3 mRNA is the most frequently and most strongly expressed subtype receptor among the SST receptors. Virgolini et al, Eur. J. Clin. Invest., 27:645-647 (1997) reports such receptors on intestinal adenocarcinomas, and neuroendocrine tumors. As a result, the peptide radiopharmaceuticals that have been developed have been found to be useful for detection and visualization of tumors bearing somatostatin receptors, and those compounds that contain a complex with 111In or 90Y are very promising radioligands for receptor-mediated radiotherapy. SSTR3 is therefore considered to be an important target both for the physiological actions of SRIF and for certain therapeutic actions of SRIF analogs.
Because of the presence of SSTR3 on tumors, and because of the otherwise ubiquitous nature of the somatostatin receptors, it would be valuable to have somatostatin analogs that would bind strongly to SSTR3 while at the same time showing only minimal propensity for binding to SSTR1-2 and SSTR4-5. The search has continued for somatostatin analogs which are more potent than somatostatin and/or exhibit dissociated inhibitory functions, and particularly for analogs which are selective for SSTR3. Nonpeptide SRIF agonists have been identified using combinatorial chemistry which exhibit selectivity for each of SSTR1 to SSTR5, Rohrer, S. P. et al., Science, 282, 737-740, Oct. 23, 1998. However, no peptide ligand has thus far been available that selectively binds to SSTR3 and exhibits fairly high affinity, as a result of which efforts to determine the precise localization of SSTR3 in the body and to identify its biological actions have been hindered; moreover, this lack of selective SSTR3 peptide ligands having relatively high affinity has hampered efforts to design more selective tumor treatment and radionuclide therapy, because only peptide ligands can be satisfactorily derivatized to incorporate complexing agents for radionuclides.
Certain modifications have now been discovered which are effective to create peptide analogs of SRIF that are selective for SSTR3 in contrast to the other cloned SRIF receptors. The preferred modification substitutes Nxcex2MeAgl(Np) into the 8-position of a SRIF analog that otherwise binds to SSTR3, and the binding strength of such analog can be enhanced by an optional modification at the N-terminus and/or in the 11-position. As a result, peptides have now been created that bind selectively to cloned SSTR3, and analogs of these peptides can be iodinated or otherwise radiolabeled while retaining their desirable biological properties. These novel peptides are useful in determining the tissue and cellular expression of the receptor SSTR3 and its biological role in the endocrine, exocrine and nervous system, as well as in regulating certain pharmacological functions without the accompanying side effects heretofore characteristic of administering SRIF. These long-acting SRIF analog peptides, when radiolabeled, can be used in scintigraphy in order to locate, i.e. localize, tumors expressing these receptors, either in vitro or in vivo; other labeling as well known in this art, e.g. fluorescent, can alternatively be used. With an appropriate chelated radioligand, these analogs can be turned into radiopharmaceuticals which are suitable for radionuclide therapy in treatment of such tumors; alternatively, they can be covalently joined to a cytotoxic moiety using an appropriate covalent conjugating agent, e.g. glutaraldehyde or one which binds via a disulfide linkage.
The SRIF analog peptides of the invention inhibit the binding of 125I-[Tyr11]SRIF and 125I-[Leu8,D-Trp22,Tyr25]SRIF-28 to the cloned human receptor SSTR3, but they do not strongly bind to SSTR1, SSTR2, SSTR4 or SSTR5. Additional of these SRIF analogs which incorporate an iodinated tyrosine in position-2 of the native molecule also do not bind to SSTR1, 2, 4 or 5 but still bind potently and saturably to SSTR3. This is also true for analogs to which 99Tc, 111In or 90Y, for example, has been chelated by linkers, such as DOTA or DTPA, or to which other complexing or conjugating agents are linked to the N-terminus for the purpose of attaching moieties useful for diagnostic or therapeutic purposes.
Many of these SRIF analogs not only selectively bind to SSTR3, but they bind thereto with high affinity. By selectively binding is meant that they exhibit a KD or an IC50 with SSTR3 which is about one-tenth or less of that with respect to at least 3 of the other five SRIF receptors. It is believed the four residues located centrally within the ring structure, i.e. at positions 7-10 of the native molecule, are primarily responsible for receptor binding and biological activity. These SRIF analogs can also be readily labeled and effectively used in drug screening methods and in radionuclide and cytotoxic therapy. For example, these analogs are useful in localizing such receptor in the body and in diagnosing the locations of tumors, particularly neuroendocrine tumors. As radionuclide therapeutic agents, they are considered to be particularly useful in combating tumors mediated by the SSTR3 receptors, as demonstrated by [90Y-DOTA-Tyr3]-Octreotide; however, they are able to accomplish this without the side effects that would otherwise accompany administration of currently available Octreotide analogs which have a propensity to interact with a plurality of SRIF receptors, i.e. SSTR2, SSTR3 and SSTR5.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. The nomenclature used to define the peptides is that specified by Schroder and Lubke, xe2x80x9cThe Peptidesxe2x80x9d, Academic Press (1965) wherein, in accordance with conventional representation, the amino group appears to the left and the carboxyl group to the right. The standard 3-letter abbreviations to identify the alpha-amino acid residues, and where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented unless otherwise expressly indicated, e.g. Ser=L-serine. By D,L is meant a mixture of the D- and L-isomers of a particular xcex1-amino acid.
SRIF analog peptides are provided having a selective affinity for the SRIF receptor SSTR3; the preferred analogs also have a high affinity for SSTR3, i.e. equal to a KD of about 10 nanomolar or less. These peptides broadly encompass known analogs of SRIF, or obvious variations thereof, which either have a D-isomer amino acid having a particular aromatic side chain in the position corresponding to the 8-position of the native peptide or have an L-isomer amino acid of this same general character in the position that corresponds to the 7-position of the native peptide. To create this particular specificity, preferably the 8-position residue should be Nxcex2MeD-Agl(Np); alternatively, the 7-position residue may be Nxcex2MeAgl(Bz). So long as the basic analog being modified exhibits SRIF properties by binding generally to SRIF receptors, insertion of such a residue in the corresponding 7- or 8-position will create a molecule which is highly selective for the SSTR3 receptor. Preferably the 1-, 2-, 4-, 5-, 12- and 13-position residues are deleted to increase binding affinity to SSTR3.
Since the characterization of SRIF, a large number of SRIF analogs have been synthesized having increased potency in some respect. The following U.S. patents are illustrative of such SRIF analogs, which analogs can be rendered selective for the SSTR3 receptor by the incorporation of the modification of the present invention: U.S. Pat. Nos. Re. 30,548; 4,133,782; 4,211,693; 4,316,891; 4,372,884; 4,393,050; 4,061,608; 4,081,433; 4,182,707; 4,190,575; 5,185,010; 4,215,039; 4,230,617; 4,238,481; 4,253,998; 4,282,143; 4,328,214; 4,358,439; 4,209,441; 4,210,636; 4,316,890; and 5,073,541.
Examples of representative peptides exhibiting the desired specificity for SSTR3 are provided by the following amino acid sequence, which is based upon a numbering system consistent with the 14-residue sequence of native mammalian SRIF, but in which the residues at positions 4-5 and 12-13 have been eliminated: (cyclo 3-14)Xaa1-Xaa2-Xaa3-Phe-Xaa7-D-Xaa8-Lys-Thr-Xaa11-Cys wherein Xaa1 is des-Xaa, Ala, D-Ala, Cbm, L-Hor, an acyl group having up to 7 carbon atoms, e.g. 4-hydroxybenzoyl, or alkyl (C1 to C6); Xaa2 is Tyr, D-Tyr, Gly or des-Xaa; -Xaa3 is Cys or D-Cys; Xaa7 is an amino acid selected from the group consisting of (A)Phe, Tyr, or Nxcex2MeAgl(Bz) wherein A is H, Cl, F, Br, NO3, Me OMe or NH(Q) where Q is H, Cbm or L-Hor; D-Xaa8 is an amino acid selected from the group consisting of D-Nal and Nxcex2MeD-Agl(Np), provided that either Xaa7 is Nxcex2MeAgl(Bz) or D-Xaa8 is Nxcex2MeD-Agl(B); and Xaa11 is Phe, Aph(X) or Tyr, with X being H, Ac or Cbm. A tyrosine residue at position 2, 7 or 11 may be radioiodinated without adversely affecting binding affinity; however, when such a radiolabelled analog is created, radioiodination of Tyr2 or Tyr11 is preferred. As previously indicated, a complexing agent can be linked to the xcex1-amino group at the N-terminal of any of these peptide analogs which is capable of joining thereto a radioactive nuclide or a cytotoxin.
One preferred subgenus of SRIF analogs has the amino acid sequence:
(cyclo 3-14)Xaa1-Xaa2-Xaa3-Phe-Xaa7-D-Xaa8-Lys-Thr-Xaa11-Cys wherein Xaa1 is Cbm; Xaa2 is Tyr, D-Tyr or des-Xaa; Xaa3 is Cys or D-Cys; Xaa7 is (A)Phe or Tyr; and D-Xaa8 is Nxcex2MeD-Agl(Np). The remaining variables are as defined hereinbefore whenever they are not specified.
Another preferred subgenus of SRIF analogs has the amino acid sequence:
(cyclo 3-14)Xaa1-Xaa2-Xaa3-Phe-Xaa7-D-Xaa8-Lys-Thr-Xaa11-Cys wherein Xaa1 is Cbm or des-Xaa; Xaa2 is Tyr, D-Tyr or des-Xaa; Xaa3 is Cys or D-Cys; Xaa7 is N62 MeAgl(Bz); and D-Xaa8 is D-Nal.
An additional preferred subgenus of SRIF analogs has the amino acid sequence:
(cyclo 3-14) Xaa2-D-Cys-Phe-Xaa7-D-Xaa8-Lys-Thr-Xaa11-Cys wherein Xaa2 is Tyr, D-Tyr or des-Xaa; Xaa7 is Phe or Tyr; D-Xaa8 is Nxcex2MeD-Agl(Np); and Xaa11 is Phe or Aph(X).
Still another preferred subgenus of SRIF analogs has the amino acid sequence:
(cyclo 3-14) Xaa1-Xaa2-D-Cys-Phe-Xaa7-D-Xaa8-Lys-Thr-Xaa11-Cys wherein Xaa1 is Cbm or des-Xaa1; Xaa2 is Tyr, D-Tyr or des-Xaa; Xaa7 is Nxcex2MeAgl(benzoyl); D-Xaa8 is D-2Nal; and Xaa11 is Phe or Aph(X).
By D-Nal is meant the D-isomer of alanine which is substituted by naphthyl on the xcex2-carbon atom. D-2Nal, wherein the attachment to naphthalene is at the 2-position on the ring structure, is preferable; however, D-1Nal is generally equivalent. Cbm stands for carbamoyl and is preferred; however, lower alkyl carbamoyl, e.g. methyl isopropyl, butyl, etc., are considered to be equivalents. By Me is meant methyl. By Bzl is meant benzyl, and by Bz is meant benzoyl. By Aph is meant aminophenylalanine, preferably 4Aph. By Ac is meant acetyl, and by Np is meant naphthoyl. By Agl is meant aminoglycine; it is always present in a form wherein the beta-amino group is alkylated with a methyl group and also acylated. It is present as the L-isomer when used in the 7-position and as the D-isomer when used in the 8-position. By Hor is meant the L-isomer of hydroorotic acid. As used herein, naphthoyl is inclusive of 1- and 2-naphthoyl, with 2-naphthoyl being preferred. By SRIF is meant the 14-residue cyclic peptide, somatostatin.
The C-terminus is usually free acid, although an equivalent, e.g. OMe or NH2, might be used. The N-terminus may be modified in various ways without significantly adversely effecting the binding affinity, all of which modifications in these cyclic peptides are considered to be included as a part of the peptides of the overall invention. For example, a variety of additions may be made to the N-terminal amino acid in the form of complexing or conjugating agents which can be then used to join a desired moiety to the peptide. For example, chelating agents, such as DTPA, DOTA, HYNIC and P2S2-COOH may be attached; alternatively, a cytotoxin may be covalently linked thereto via a conjugating agent if desired. When either Tyr or D-Tyr appears at the N-terminus, it may be in the xe2x80x9cdesaminoxe2x80x9d form and/or may be radioiodinated or otherwise labeled. Acyl groups having not more than about 20 carbon atoms, e.g. 4-hydroxybenzyl, may also be present at the N-terminus, as bulky moieties appear to be accommodated without loss of selectivity.
Although SSTR3 was one of the first somatostatin receptors cloned, identification of its biological and pharmacological properties has lagged somewhat behind the other SRIF receptors because of the lack of ligands which are significantly selective for SSTR3. The peptides of the invention are believed to be the first truly SSTR3-selective peptides, and for a number of reasons it is considered advantageous to have peptide, rather than nonpeptide, ligands of this character. They will be very helpful in determining the many functional roles of this receptor and in selectively binding only this SRIF receptor and not the others, and they will be particularly valuable in SRIF receptor-targeted scintigraphy and radionuclide therapy.
Selectivity for binding of the analog peptides of the invention to SSTR3 was demonstrated by testing their interaction with the five different cloned human SRIF receptors as described in great detail hereinafter. Generally, recombinant cells expressing the receptor are washed and homogenized to prepare a crude protein homogenate in a suitable buffer, as known in the art. In a typical assay, an amount of protein from the cell homogenate is placed into a small volume of an appropriate assay buffer at an appropriate pH. Candidate substances, such as potential SRIF agonists and antagonists, are added to the admixture in convenient concentrations, and the interaction between the candidate substance and the receptor polypeptide is monitored. The peptides of the invention bind substantially only to SSTR3, and their binding exhibits high affinity.
Receptor binding assays are performed on cloned SRIF receptors as generally set forth in Raynor et al. supra. Using such assays, one can generate KD values which are indicative of the concentration of a ligand necessary to occupy one-half (50%) of the binding sites on a selected amount of a receptor or the like, or alternatively, competitive assays can generate IC50 values which are indicative of the concentration of a competitive ligand necessary to displace a saturation concentration of a target ligand being measured from 50% of binding sites. The peptide des-AA1,2,4,5,12,13-[D-Agl8(Me,2-naphthoyl)]SRIF inhibits the binding to SSTR3 of an iodinated SRIF-28 ligand that has strong affinity for all five receptors. Testing shows that it binds to the cloned human SSTR3 with an IC50 of about 70 nM, while this SRIF analog peptide does not bind to human SSTR1, SSTR2, or SSTR5 at concentrations below 10,000 nM nor to SSTR4 at a concentration below 1,000 nM.
When this SRIF analog is modified to have a tyrosine residue in position-2 which is then radioiodinated, testing for binding to the cloned human SRIF receptors shows the 125I-Tyr2 analog likewise did not bind to SSTR1, 2, 4 or 5, but continues to bind saturably to SSTR3. Another exemplary analog, des-AA1,2,4,5,12,13-[Agl7(Me,Bz), D-2Nal8]-SRIF inhibits binding of the same iodinated SRIF-28 ligand to SSTR3 and binds itself with an IC50 of about 80 nM, while not binding to any of the other 4 receptors at concentrations below 1000 nM. These SRIF analogs that selectively show high affinity to SSTR3 are considered to be particularly useful in combating tumors by carrying radionuclides or cytotoxins to the sites of these receptors but not to other SRIF receptors.
As hereinbefore indicated, SSTR3 mRNA has been detected in a variety of tumors. However, it is presently not known whether SSTR3 plays a major role in tumor growth regulation and, if it does, whether it mediates simulation or inhibition. Therefore, it is difficult to foretell whether a selective SSTR3 antagonist would have a beneficial role for long-term treatment of tumors. However, the use of SRIF analogs selective for SSTR3 that bind strongly thereto, and that are long-acting can be effectively used to kill such tumors via radionuclide or cytotoxic therapy. To date the use of Octreotide in the treatment of such tumors has not been considered to be satisfactorily effective.
Although an analog of Octreotide has been employed to detect human tumors having high expression of SRIF receptors through the use of positron-emission tomography, this SRIF analog does not distinguish among SSTR2, SSTR3 and SSTR5. In comparison, radiolabeled SRIF analogs of the present invention can be employed for similar purposes, and they are considered to be specifically useful in identifying tumors expressing SSTR3, which tumors are then therapeutic targets for treatment with SSTR3-selective ligands as mentioned hereinbefore.
The SRIF analogs of the present invention are the first peptide analogs truly selective for SSTR3 and are considered to be useful in combating cancers which express SSTR3. They are also considered to be most useful in scintigraphy to determine the distribution of cells and tissues expressing this receptor in the brain and in the endocrine and exocrine systems, and also in identifying selective functions of this receptor in the body. They are further useful for treating non-neoplastic disorders linked to SSTR3-expressing tissues, such as have been found in the GI track smooth muscles. In other words, SSTR3 antagonists may be useful to treat gastrointestinal motility disorders.
Labeled SRIF analogs of the invention are also considered to be useful in drug-screening assays to screen for new effective peptide and nonpeptide agents which will bind with high affinity to SSTR3 and which may be either highly effective agonists or antagonists for treating GI track motility. Once a known ligand for the receptor SSTR3 is in hand, one can obtain a baseline activity for the recombinantly produced receptor. Then, to test for inhibitors or modifiers, i.e. antagonists of the receptor function, one can incorporate into a test mixture a candidate substance to test its effect on the receptor. By comparing reactions which are carried out in the presence or absence of the candidate substance, one can then obtain information regarding the effect of the candidate substance on the normal function of the receptor. The cyclic SRIF analogs described in Examples 2-2E hereinafter are antagonists and can be employed to selectively inhibit the normal function of SSTR3.
The peptides of the present invention can be synthesized by classical solution synthesis, but they are preferably synthesized by solid-phase technique. A chloromethylated resin or a hydroxymethylated resin is preferably used. For example, these peptides having a free carboxyl C-terminus are preferably synthesized as taught in U.S. Pat. No. 4,816,438 issued Mar. 28, 1989, the disclosure of which is incorporated herein by reference. Solid-phase synthesis is conducted in a manner to stepwise add amino acids in the chain beginning at the C-terminus in the manner set forth in that U.S. patent. Side-chain protecting groups, which are well known in the art, are preferably included as a part of any amino acid which has a particularly reactive side chain, and optionally may be used in the case of others such as Trp, when such amino acids are coupled onto the chain being built upon the resin. Such synthesis provides the fully protected intermediate peptidoresin.
Chemical intermediates which are used to synthesize certain preferred SRIF analogs may be represented by the formula:
X1-Xaa2(X2)-Cys(X3)-Phe-Xaa7-D-Xaa8-Lys(X4)-Thr(X5)-Xaa11-Cys(X3)-X6 wherein
X1 is an xcex1-amino protecting group of the type known to be useful in the art in the stepwise synthesis of a polypeptide, e.g. tertbutyloxycarbonyl(Boc).
X2 is hydrogen or a protecting group for the phenolic hydroxyl group of Tyr, such as 2-bromobenzyloxycarbonyl (2BrZ).
X3 is hydrogen or a protecting group for Cys, such as p-methoxybenzyl(Mob) or acetamidomethyl (Acm).
X4 is a protecting group for an amino side chain group, such as 2-chlorobenzyloxycarbonyl(2Cl-Z), which is not removed during deprotection of the xcex1-amino groups during the synthesis.
X5 is hydrogen or a protecting group for the hydroxyl side chain of Thr or Ser, e.g. benzyl ether(Bzl).
X6 is selected from the class consisting of OH, OCH3 and esters, including a benzyl ester or a hydroxymethyl ester anchoring bond used in solid-phase synthesis for linking to a solid resin support, such as
xe2x80x94Oxe2x80x94CH2-polystyrene resin support.
Thus, there is broadly provided a method for making a SRIF analog peptide having the formula:
(cyclo 3-14)H-Xaa2-Cys-Phe-Xaa7-D-Xaa8-Lys-Thr-Xaa11-Cys-OH
wherein Xaa2, Xaa7 and D-Xaa8 are as set forth hereinbefore, by first forming an intermediate peptide having the formula:
X1-Xaa2(X2)-Cys(X3)-Phe-Xaa7-D-Xaa8-Lys(X4)-Thr(X5)-Xaa11-Cys(X3)-X6
and then splitting off any protecting groups X1 to X5 and/or cleaving from any resin support included in X6 before oxidizing to create a disulfide bond between the Cys side chains.
The SRIF analogs of the invention are generally effective at levels of less than 100 micrograms per kilogram of body weight. For prolonged action, it may be desirable to use dosage levels of about 0.1 to about 2.5 milligrams per kilogram of body weight. These analogs are soluble in water and thus can be prepared as relatively concentrated solutions for administration.